Mechanism of total triterpenoids from fruits of Chaenomeles speciosa in alleviating N-methyl-N′-nitro-N-nitrosoguanidine and nigericin induced GES-1 cell damage based on miR-210/NF-κB/NLRP3 inflammasome signaling pathway
ZHANG Jihong, ZHOU Jiangpeng, MA Junhui, GAN Tingyue, HE Haibo, ZHOU Rufeng, YU Huifan, LI Haoran, WU Mian, XU Jie, PAN Chaowang
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doi: 10.7501/j.issn.0253-2670.2026.12.013
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目的 基于微小RNA-210（microRNA-210，miR-210）/核因子-κB（nuclear factor-κB，NF-κB）/NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）炎症小体信号通路研究木瓜总三萜（total triterpenes from fruits of Chaenomeles speciosa，CST）减轻N-甲基-N′-硝基-N-亚硝基胍（N-methyl-N′-nitro-N-nitrosoguanidine，MNNG）和尼日利亚菌素（nigericin，NIG）诱导人胃黏膜上皮细胞损伤的作用机制。方法 构建MNNG和NIG损伤GES-1细胞模型，使用CST和（或）miR-210 mimic、miR-210 inhibitor、NF-κB siRNA、NLRP3 siRNA处理GES-1细胞。采用MTT法检测细胞活力；免疫荧光检测细胞内活性氧（reactive oxygen species，ROS）水平；流式细胞术检测细胞焦亡；比色法和ELISA法检测细胞上清液中白细胞介素-4（interleukin-4，IL-4）、IL-1β、IL-6、IL-10、IL-18、乳酸脱氢酶（lactate dehydrogenase，LDH）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平；比色法检测细胞中过氧化氢酶（catalase，CAT）、谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）、髓过氧化物酶（myeloperoxidase，MPO）、超氧化物歧化酶（superoxide dismutase，SOD）、总抗氧化能力（total antioxidant capacity，T-AOC）水平；双荧光素酶报告实验验证miR-210与NF-κB的靶向关系；采用免疫荧光检测NF-κB p65核移位及NLRP3、凋亡相关斑点样蛋白（apoptosis related spot like protein，ASC）和半胱氨酸天冬氨酸蛋白酶-1前体（pro-cystein-asparate protease-1，pro-Caspase-1）共定位；qRT-PCR测定miR-210、NF-κB、NLRP3 mRNA表达；Western blotting检测胞质NF-κB p65、胞核NF-κB p65、细胞有丝分裂相关酶7（NIMA-related kinase 7，NEK7）、硫氧还蛋白互作蛋白（thioredoxin interacting protein，TXNIP）、NLRP3、ASC、Caspase-1、pro-Caspase-1、消皮素D（gasdermin D，GSDMD）、GSDMD的N端结构域（N-terminal domain of GSDMD，GSDMD-N）、pro-IL-18、pro-IL-1β蛋白表达。结果 miR-210是促炎/促焦亡因子，可与NF-κB mRNA 3’-UTR结合。与模型组比较，CST可显著抑制MNNG和NIG所致的GES-1细胞焦亡和LDH释放（P＜0.01），降低细胞上清液中IL-1β、IL-6、IL-18、TNF-α和细胞中ROS、MPO、MDA水平（P＜0.01），升高细胞上清液中IL-4、IL-10水平和细胞中CAT、GSH、SOD、T-AOC活性（P＜0.01）；抑制NF-κB p65核移位及NLRP3、ASC和pro-Caspase-1共定位（P＜0.01）；显著下调细胞中miR-210、NF-κB、NLRP3 mRNA和胞核NF-κB p65、TXNIP、NEK7、NLRP3、ASC、pro-Caspase-1、Caspase-1、GSDMD、GSDMD-N、pro-IL-1β、pro-IL-18蛋白表达（P＜0.01），上调胞质NF-κB p65蛋白表达（P＜0.01）。CST与miR-210 inhibitor、NF-κB siRNA、NLRP3 siRNA转染联用可进一步改善上述指标（P＜0.01）。结论 CST对MNNG和NIG诱导的GES-1细胞损伤具有较好的防治作用，其机制与抑制miR-210异常升高和NF-κB/NLRP3炎症小体信号通路激活、减轻氧化应激，进而抑制细胞焦亡密切相关。
Objective To investigate the mechanism of total triterpenes from fruits of Chaenomeles speciosa (CST) in alleviating N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and (NIG)-induced human gastric mucosal epithelial cells damage based on microRNA-210 (miR-210)/nuclear factor-κB (NF-κB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome signaling pathway. Methods MNNG and NIG induced GES-1 cells model were constructed, and then GES-1 cells were treated with CST and/or miR-210 mimic, miR-210 inhibitor, NF-κB siRNA, NLRP3 siRNA. MTT assay was used to detect cell viability, immunofluorescence was utilized to measure reactive oxygen species (ROS) level, flow cytometry was used to test pyroptosis, colorimetric and ELISA methods were utilized to measure the levels of interleukin-4 (IL-4), IL-1β, IL-6, IL-10, IL-18, lactate dehydrogenase (LDH), and tumor necrosis factor-α (TNF-α) in cell supernatant. The colorimetric method was used to determine the levels of catalase (CAT), glutathione (GSH), malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in cells. The dual luciferase reporter assay was employed to verify the targeted intervention between miR-210 and NF-κB. Immunofluorescence was utilized to detect the NF-κB p65 nuclear translocation, the co-localization of NLRP3, apoptosis related spot like protein (ASC) and pro-cystein-asparate protease-1 (pro-Caspase-1). qRT-PCR was used to detect the mRNA expression levels of miR-210, NF-κB, NLRP3. Western blotting was utilized to determine the protein expression levels of cytoplasmic NF-κB, nuclear NF-κB, NIMA-related kinase 7 (NEK7), thioredoxin-interacting protein (TXNIP), NLRP3, ASC, Caspase-1, pro-Caspase-1, gasdermin D (GSDMD), N-terminal domain of GSDMD (GSDMD-N), pro-IL-18 and pro-IL-1β. Results miR-210 was a pro-inflammatory/pyroptosis factor, the dual luciferase reporter assay confirmed that it could bind to 3’-UTR in mRNA of NF-κB. Compared with model group, CST significantly inhibited pyroptosis and LDH release in MNNG and NIG induced GES-1 cells (P < 0.01), reduced levels of IL-1β, IL-6, IL-18, TNF-α in supernatant and ROS, MPO, MDA in cells (P < 0.01), elevated levels of IL-4, IL-10 in supernatant and activities of CAT, GSH, SOD, T-AOC in cells (P < 0.01), suppressed the NF-κB p65 nuclear translocation and the co-localization of NLRP3, ASC and pro-Caspase-1 (P < 0.01), downregulated the mRNA expressions of miR-210, NF-κB, NLRP3 and the protein expressions of nuclear NF-κB p65, TXNIP, NEK7, NLRP3, ASC, pro-Caspase-1, Caspase-1, GSDMD, GSDMD-N, pro-IL-1β, pro-IL-18 (P < 0.01), upregulated the cytoplasmic NF-κB p65 protein expression (P < 0.01). The combination of CST with miR-210 inhibitor, NF-κB siRNA and NLRP3 siRNA transfection could further improve the above indicators (P < 0.01). Conclusion CST has a good preventive and therapeutic effect on MNNG and NIG-induced GES-1 cell injury, and its mechanism is closely related to inhibiting the abnormal elevation of miR-210 and activation of NF-κB/NLRP3 inflammasome signaling pathway, alleviating oxidative stress and subsequent suppressing pyroptosis.
Key words
total triterpenes from fruits of Chaenomeles speciosa
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inflammatory response
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oxidative stress
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pyroptosis
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miR-210/NF-κB/NLRP3 inflammasome signaling pathway
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chronic atrophic gastritis
ZHANG Jihong, ZHOU Jiangpeng, MA Junhui, GAN Tingyue, HE Haibo, ZHOU Rufeng, YU Huifan, LI Haoran, WU Mian, XU Jie, PAN Chaowang.
Mechanism of total triterpenoids from fruits of Chaenomeles speciosa in alleviating N-methyl-N′-nitro-N-nitrosoguanidine and nigericin induced GES-1 cell damage based on miR-210/NF-κB/NLRP3 inflammasome signaling pathway[J].
Chinese Traditional and Herbal Drugs,
2026
, 57
(12)
: 4662
-4683
.
DOI: 10.7501/j.issn.0253-2670.2026.12.013
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